摘要
As a robust platform for genome editing,CRISPR/Cas9 is currently being explored for engineering biology or therapeutics,yet means for quantitative detection of Cas9 proteins remain to be fully realized.Here,we expressed Cas9 proteins and developed a novel detection method that traced Cas9 based on radiolabeled iodine.Through optimizing the reaction conditions of reaction time,temperature and cycles,we obtained ^(125)I-Cas9 of high labeling yield.The prepared ^(125)I-Cas9 was stable in various media and preserved excellent genome editing efficiency.Thus,our strategy provides a convenient and efficient tool for further tracing biological behaviors of Cas9 proteins in living systems.
As a robust platform for genome editing, CRISPR/Cas9 is currently being explored for engineering biology or therapeutics, yet means for quantitative detection of Cas9 proteins remain to be fully realized. Here, we expressed Cas9 proteins and developed a novel detection method that traced Cas9 based on radiolabeled iodine. Through optimizing the reaction conditions of reaction time, temperature and cycles, we obtained <sup>125</sup>I-Cas9 of high labeling yield. The prepared <sup>125</sup>I-Cas9 was stable in various media and preserved excellent genome editing efficiency. Thus, our strategy provides a convenient and efficient tool for further tracing biological behaviors of Cas9 proteins in living systems.
基金
supported by the National Key Research and Development Program(No.2016YFA0400902)
the Ministry of Science and Technology of China(Nos.2012CB825805,2012CB932600)
the National Natural Science Foundation of China(Nos.11675251 and 11275251)
Shanghai Rising-Star Program(No.14QA1404400)
Distinguished Scientist Fellowship Program of King Saud University
the Youth Innovation Promotion Association of CAS(No.2016236)
